The canonical Wnt/β-catenin pathway is of particular importance in regulating cell proliferation, differentiation and cell-cell communication. The aberrant activation of Wnt/β-catenin signaling leads to the initiation and progression of many cancers such as colorectal cancers (P. Morin, et al. Science 275(1997) 1787-1790), hepatocellular carcinoma (A., de La Coste, et al. Proc. Natl. Acad. Sci. U.S.A. 95(1998) 8847-8851), breast cancers (C. Scheel, E. N. Eaton, S. H. Li, et al. Cell 145(2011) 926-940), leukemia (D. Lu, et al. Proc. Natl. Acad. Sci. U.S.A. 101(2004) 3118-3123), and multiple myeloma (K. Sukhdeo, et al. Proc. Natl. Acad. Sci. U.S.A. 104(2007) 7516-7521). Moreover, cancer stem cells, which are resistant to conventional chemotherapies and are especially virulent, are controlled by the overactivated Wnt//β-catenin signaling (L. Vermeulen, E. De Sousa, F. Melo, et al. Nat. Cell Biol. 2010, 12(5), 468-476; C. Scheel, E. N. Eaton, S. Li, et al. Cell 2011, 145(6), 926-940). In addition, dysfunction in the Wnt/β-catenin signaling pathway can lead to fibrotic diseases, e.g. pulmonary fibrosis (W. R. Henderson Jr., et al. Proc. Natl. Acad. Sci. U.S.A. 107(2010) 14309-14314), liver fibrosis (J. H. Cheng, et al. Am. J. Physiol. Gastrointest. Liver Physiol. 294(2008) G39-G49) and cystic kidney disease (M. A. Lancaster, et al. Nat. Med. 15(2009) 1046-1054).
β-catenin is the key mediator of the canonical Wnt pathway. It is tightly controlled by a multi-protein destruction complex composed of scaffolding protein Axin, tumor suppressor adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 3β(GSK3β), which phosphorylates β-catenin and induces its ubiquitination and proteasomal degradation (H. Clevers. Cell 127(2006) 469-480). The inhibition of β-catenin degradation in the pathological conditions, usually through the mutation of genes that encode Axin, APC or the N-terminal phosphorylation sites of β-catenin, results in the stabilization and nuclear translocation of cytoplasmic β-catenin (P. Polakis. Curr. Opin. Genet. Dev. 17 (2007) 45-51). In the cell nucleus, β-catenin forms a complex with the N-terminus of T-cell factor (Tcf)/Lymphoid enhancer factor (Lef), and activates the transcription of Wnt target genes, such as c-myc, cyclin D1 and survivin, which lead to the initiation and proliferation of cancer cells, fibrotic cells, and/or the self-renewal of cancer stem cells.
The transcriptional over-activation of Wnt/β-catenin target genes is solely dependent on the formation of nuclear β-catenin/Tcf complex. Thus, selective inhibition of β-catenin/Tcf protein-protein interactions is a potentially appealing therapeutic strategy. Despite significant efforts directed to determination of small-molecule inhibitors of β-catenin/Tcf interactions, little progress had been made to date and only four examples have been reported.
Shivdasani and co-workers screened libraries of synthetic and natural compounds and identified six natural products as the inhibitors of β-catenin/Tcf protein-protein interactions. Three compounds were isolated from fungal organisms, and are planar multi-ring perylenequinones, PKF115-584, CGP049090 and PKF222-815. Three compounds were isolated from the Actinomycete strains, ZTM000990, PKF118-310 and PKF 118-744. The IC50 values of these compounds in an ELISA-based β-catenin/Tcf interaction assay are between 0.64 and 8.7 μM (M. Lepourcelet, Y. P. Chen, D. S. France, et al. Cancer Cell 5(2004) 91-102). Two compounds, PKF115-584 and CGP049090 (shown in FIG. 1) have been reported to inhibit Tcf reporter activity, block the expression of Wnt target genes and the growth of colon cancer cells. Subsequent studies suggested that these two compounds, PKF 115-584 and CGP049090, selectively induced the apoptosis of primary acute myeloid leukemia (AML) (K. S. Minke, P. Staib, A. Puetter, et al. Eur. J. Haematol. 82 (2009) 165-175), adrenocortical carcinoma (M. Doghman, J. Cazareth, E. Lalli. J. Clin. Endocrinol. Metab. 93 (2008) 3222-3225), B-cell chronic lymphocytic leukemia (CLL) (R. K. Gandhirajan, P. A. Staib, K. Minke, et al. Neoplasia 12(2010) 326-335) and hepatocellular carcinoma (HCC) (W. Wei, M. S. Chua, S. Grepper, et al. Int. J. Cancer. 126(2010) 2426-2436). In the xenograft models of these two compounds, PKF 115-584 and CGP049090, suppress the growth of tumors and prolong survival, e.g. xenografts of human multiple myeloma (K. Sukhdeo, M. Mani, Y. Zhang, et al. Proc. Natl. Acad. Sci. U.S.A. 104(2007) 7516-7521) and hepatocellular carcinoma (W. Wei, M. S. Chua, S. Grepper, et al. Int. J. Cancer. 126(2010) 2426-2436).
A combination of virtual screening, WaterLOGSY NMR and isothermal titration calorimetry (ITC) studies identified PNU-74654 (shown in FIG. 1; see J. Trosset, C. Dalvit, S. Knapp, et al. Proteins 64(2006) 60-67). The dissociation constant (Kd) of this compound with β-catenin is 0.45 μM by ITC. DasGupta and co-workers developed an RNAi-based high-throughput screening method to identify inhibitors of β-catenin-responsive transcription (F. C. Gonsalves, K. Klein, B. B. Carson, et al. Proc. Natl. Acad. Sci. U.S.A. 108(2011) 5954-5963.), which they used to screen small-molecule libraries. This cell-based assay was designed to identify inhibitors of the Wnt cascade downstream of the multi-protein destruction complex composed of scaffolding protein Axin, tumor suppressor adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 33 (GSK3β). Three compounds, iCRT3, iCRT5 and iCRT14, were identified in this assay system (shown in FIG. 1). A protein pulldown assay using GST (26 kD)-fused human Tcf4 (residues 1-55, 5.8 kD) and β-catenin revealed that these compounds, iCRT3, iCRT5 and iCRT 14, disrupted β-catenin/Tcf interactions, and inhibited the transcription of Wnt target genes and the growth of colon cancer cells.
Recently, An and coworkers identified an organic copper compound, BC21 (NCI-109268; shown in FIG. 1), as an inhibitor of β-catenin/Tcf interaction by virtual screening and a luciferase-based reporter gene assay. The activity of this compound was confirmed by a fluorescence polarization assay using the shorter peptide segment of human Tcf4 (residue 8-30) (W. Tian, X. Han, M. Yan, et al. Biochemistry (2012), ASAP. DOI: 10.1021/bi201428h). However, the known inhibitors of β-catenin/Tcf protein-protein interactions discussed above are either not good drug candidates (PKF 115-584, CGP049090 and BC21), or possess weak activity (PNU-74654, iCRT3, iCRT5 and iCRT14).
WO 98/42296 discloses purified β-catenin proteins and conventional heterogeneous bioassays, such as ELISA, to screen inhibitors of β-catenin/Tcf interactions. WO 01/19353A2 discloses potential “targetable” pockets that maya be responsible for β-catenin/Tcf4 interactions. Also described therein is a in silico screening algorithm and inhibitors were identified by an ELISA screening. WO 02/44378A2 discloses a luciferase reporter gene assay for screening inhibitors of β-catenin/Tcf interaction. WO02/096430A1 discloses cephalosoporin derivatives as possible inhibitors to disrupt β-catenin/Tcf interaction. WO 03/006447A2 discloses PNU-74654 and related compounds identified via a combination of virtual screening, WaterLOGSY NMR and isothermal titration calorimetry (ITC) studies. US 2004/0005313A1 and US 2006/0165699A1 describes the use of microarray technology, Northern blot and RT-PCR analyses of a group of target genes regulated by β-catenin/Tcf complexes, and used this information to identify possible inhibitors. WO 2008/147713A1 discloses debromohymenialdesine (dBHD) and related derivatives as inhibitors of β-catenin/Tcf protein-protein interactions. JP2008/214243A discloses aureothin as an inhibitor of β-catenin/Tcf interaction. WO 2009/097113A2 describes the compounds iCRT3, iCRT5, and iCRT14, as well as related derivative, that were identified through an RNAi-based high-throughput screenings. WO 2010/014043A1 discloses that RUNX3 forms a ternary complex with the β-catenin/Tcf complex and attenuates the Wnt signaling. The phosphorylation of RUNX3 is reported to diminish the formation of the ternary complex, thus increasing Wnt signaling. The inhibition of RUNX3 phosphorylation by inhibitors is a possible therapeutic approach targeting this pathway. U.S. Pat. No. 7,999,089B1 describes RNA aptamers that bind to β-catenin and disrupt the β-catenin/Tcf complex.
Despite advances in research directed to identifying inhibitors the Wnt signaling pathway generally, and specifically inhibitors of β-catenin/Tcf interactions, there remains a scarcity of compounds that are both potent, efficacious, and selective inhibitors of β-catenin/Tcf interactions and also effective in the treatment of cancers and other diseases associated with uncontrolled cellular proliferation, e.g. fibrotic diseases, associated with β-catenin/Tcf dysfunction. These needs and other needs are satisfied by the present invention.